A recent study has shown that laser causes an increase in equine superficial tissue temperature, which may result in an increase in blood perfusion and a stimulating effect on tissue reg
Trang 1A Bergh 1 , G Nyman 2 , T Lundeberg 3 and S Drevemo 1 : Effect of defocused CO 2
laser on Equine Tissue perfusion Acta vet scand 2006, 47, 33-42 – Treatment with
defocused CO2laser can have a therapeutic effect on equine injuries, but the
mecha-nisms involved are unclear A recent study has shown that laser causes an increase in
equine superficial tissue temperature, which may result in an increase in blood perfusion
and a stimulating effect on tissue regeneration However, no studies have described the
effects on equine tissue perfusion The aim of the present study was to investigate the
effect of defocused CO2laser on blood perfusion and to correlate it with temperature in
skin and underlying muscle in anaesthetized horses Differences between clipped and
unclipped haircoat were also assessed Eight horses and two controls received CO2laser
treatment (91 J/cm 2 ) in a randomised order, on a clipped and unclipped area of the
ham-string muscles, respectively The significant increase in clipped skin perfusion and
tem-perature was on average 146.3±33.4 perfusion units (334%) and 5.5±1.5 °C,
respec-tively The significant increase in perfusion and temperature in unclipped skin were
80.6±20.4 perfusion units (264%) and 4.8±1.4 °C No significant changes were seen in
muscle perfusion or temperature In conclusion, treatment with defocused CO2laser
causes a significant increase in skin perfusion, which is correlated to an increase in skin
temperature.
equine; CO 2 laser therapy; therapeutic heat; blood perfusion; laser Doppler
flowme-try; temperature; rehabilitation.
Effect of Defocused CO 2 Laser on Equine Tissue Perfusion
By A Bergh 1 , G Nyman 2 , T Lundeberg 3 and S Drevemo 1
1 Department of Anatomy and Physiology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2 De-partment of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden, and 3 Rehabilita-tion Medicine University Clinic, Stockholm, Sweden.
Introduction
The goal of physical therapy is to promote
heal-ing of tissues through stimulation of normal
physical processes, thereby restoring the
func-tion of the injured tissue (Stashak 1987)
Ther-apeutic modalities in the form of hot packs,
therapeutic ultrasound, and lasers have been
ad-vocated by numerous practitioners working
with sports injuries, both in humans and horses
(Lehmann 1990, Bromiley 1991) Simplified,
laser therapy can be divided into surgical lasers
(i.e high effect laser) and lasers used for
biomodulation, i.e low level laser therapy
(of-ten treatment dosages of <1 to 4 J/cm2on
treat-ment sites) (Basford 1995) However, lasers
originally made for surgery are used as biomod-ulating lasers; with a defocused beam and at a lower output effect, but with doses higher than
in low laser therapy A recent prospective study indicates that, defocused carbon dioxide (CO2) laser may be an applicable treatment for acute
synovitis in horses (Lindholm et al 2002) The
effects of laser radiation on tissue structure and function are, however, unclear Laser therapy is proposed to produce photochemical effects by excitation of electronic states in molecules and
laser is also proposed to have photothermal ef-fects, by transformation of absorbed light
Trang 2en-ergy to heat (Thomsen 1991) The light is
ab-sorbed at a depth of less than 0.1 mm (Bhatta
1994), which indicates a possible heating effect
in superficial tissues
Results from our own studies show that
treat-ment with defocused CO2laser causes a
signif-icant increase in the temperature of skin and
subcutis (Bergh et al 2005) A rise in local
tem-perature generally correlates with an increase in
perfusion, and is believed to have a positive
ef-fect on pain and tissue regeneration (Lehmann
1990, Nannemann 1991, Oosterveld and
Rasker 1994, Wright and Sluka 2001, Nadler et
al 2002) Vasodilatation increases blood flow
to reduce ischemia of injured tissue, resulting in
decreased activity of the pain receptors A
greater blood flow increases the supply of
nutri-ents to the area for the repair process and
re-moves by-products from the injured tissue To
the best of our knowledge, there are no studies
published on the effect of defocused CO2laser
on equine tissue perfusion
Laser Doppler Flowmetry (LDF) technique is
widely used for measurement of tissue
perfu-sion (Norman et al 1992, Adair et al 1994,
Raisis et al 2000a, Berardesca et al 2002,
Ed-ner et al 2002, McGorum et al 2002) This
technique provides a continuous measure of
rel-ative perfusion, allowing detection of changes
in blood flow over time on a single site (Raisis
et al 2000b, Raisis et al 2000c, Humeau et al.
2004) In the present study, a hypothesis was
formulated that treatment with defocused CO2
laser increases temperature and perfusion in
skin and underlying muscle The objective was
to measure temperature and perfusion in
anaes-thetized horses treated with active or sham
laser A further aim was to compare the effect of
laser treatment on clipped and unclipped skin
Materials and Methods
Horses
The study comprised ten, healthy Standardbred trotters (6 females and 4 geldings) with a mean weight of 497 kg (range 411-578 kg) and a mean age of nine years (range 4-19 years) Eight horses received laser treatment and two served as controls All horses were pigmented
at the irradiated area The Ethical Committee
on Animal Experiments in Uppsala, Sweden has approved the study
Anaesthetic protocol
Food was withheld for 12 hours prior to anaes-thesia, but water was available until premedica-tion The horses were premedicated with
Sollentuna, Sweden) Anaesthesia was induced intravenously with guaifenesin (Myolaxin® vet diluted to 7.5%; Chassot & Cie AG, Berne, Switzerland) and tiopentone (Penthotal Na-trium 12.5%; Abbott, Solna, Sweden) The horses were intubated, transported to the surgi-cal table and placed in dorsal recumbency Anaesthesia was maintained with isoflurane (Forene; Abbott, Solna, Sweden) in oxygen Electrolytes (Ringer acetate; Pharmacia & Up-john, Stockholm, Sweden) were infused through a catheter in the jugular vein Sponta-neous breathing was allowed from a semiclose, large-animal circle To detect any changes in depth of anaesthesia, arterial blood pressure as well as heart rate was monitored throughout the research protocol
Peripheral perfusion and muscle temperature
Laser Doppler Flowmetry (LDF) was per-formed using a Periflux 4001 flowmeter (Per-imed, Järfälla, Sweden) A treatment area of 6x7 cm on each semimembranosus muscle was prepared; one side clipped and the other with the coat intact A small area for the measuring probes was prepared, in direct contact with each
Trang 3treated area Skin perfusion was measured on
the skin surface (Probe 407, Perimed, Järfälla,
Sweden), 1 and 3 cm from the treated area
For muscle perfusion, a straight microtip with
slanted tip (MT A500-0.120 mm, 0.5 mm
di-ameter, Perimed, Järfälla, Sweden) was placed
in the semimembranosus muscle of the right
and left hind limb, close to the skin perfusion
probe at 1 cm from the treatment area The
mi-crotip was inserted via a 0.7 mm cannula to a
depth of 3 cm and connected to a probe (Master
Probe; Probe 418-x, Perimed, Järfälla,
Swe-den), after which the cannula was retracted
Skin and muscle temperatures were measured
using thermistor probes (skin-440,
muscle-442-PI, Perimed, Järfälla, Sweden) connected to a
recording unit (PF 5020, Perimed, Järfälla,
Sweden) The temperature probes were
at-tached to the skin or inserted in the muscle
ap-proximately 1 cm from its corresponding
perfu-sion probe and at the same distance from the
irradiated area
Flux, expressed in blood perfusion units, and
temperature, were displayed and recorded
con-tinuously (Perisoft 1; 14, Perimed, Järfälla,
Sweden) The total recording time was 50 min
on average To allow comparison of results, the
LDF probes were calibrated in a standard
motil-ity solution provided by the manufacturer The
following skin and muscle blood flow and
tem-perature features were analysed:
1 The average value before treatment, sampled for one minute (baseline)
2 The average value during treatment, sampled for one minute (treatment)
3 The peak value during treatment (peak)
4 The time from start of treatment to the peak value, in seconds (time to peak)
Laser protocol
A defocused CO2laser (10 600 nm, KSV 25S; EL.EN SRL, Firenze, Italy) was used in the study, see Table 1 As guiding light, a HeNe source (633 nm) emitted continuously at 1.2
mW The laser system was calibrated regularly, and an external detector (LaserMate Detector, COA-33-0191-000; Gamma Optronic AB, Up-psala, Sweden) was used to measure the inten-sity of the laser beam before and after each treatment The laser system was set to give con-tinuous output power of 16 W at a distance of 1
m, during a treatment period of 4 min The treatment area was 42 cm2(6x7 cm) and the ir-radiation energy 91 J/cm2 Laser-treated and control horses, as well as the order of the treat-ment to the clipped and unclipped area, were randomized There was an average of a 60-min pause between the irradiation of the clipped and unclipped area
Ta bl e 1 Laser parameters, dosage, and mode of application (KSV 25S Laser device)
Site of application semimembranosus muscle semimembranosus muscle
Trang 4Statistical analysis
Statistica 6.0 (Statsoft, 2001; Statsoft
Scandi-navia AB, Uppsala, Sweden) was used for data
analysis, and results are presented as means and
standard errors (SE) Microvascular perfusion
and temperature were calculated as the average
of one minute of stable recordings; immediately
before start of treatment, at the end of
treat-ment, and at 4 min after the end of treatment
The perfusion data are presented as relative
changes in perfusion, using arbitrary perfusion
units (PU) The data were individually
cor-rected by setting the baseline before treatment
to 100% Biological zero (i.e the laser Doppler
signal from non-perfused tissue) was not
sub-tracted; however, perfusion values under 3.5 PU
were excluded from the analysis as the
biologi-cal zero for equines is approximately 1.6 PU for
skin and 3.5 PU for muscle (unpublished
re-sults)
Treatment data were compared to baseline data
within each group Statistical calculations
com-paring the time to maximum (peak) values and
peak values for both skin temperature and
per-fusion were also made Statistical calculations
were performed with Wilcoxon signed rank test and Mann-Whitney test, when appropriate Sta-tistical significance was accepted at p<0.05
Results
None or only minor differences in arterial blood pressure or heart rate were found within each protocol or between treatment and control The results are presented separately for treated and control horses Figure 1 shows representa-tive temperature and perfusion curves from one laser and control recording, respectively As demonstrated by the laser recording, the in-crease in temperature is almost immediately followed by an increase in perfusion
The laser- treated group Temperature
The temperature response to laser treatment is presented in Table 2 The results presented for temperatures response of clipped skin, 1 cm and unclipped skin, 3 cm are from seven horses,
as measurements from one horse had to be ex-cluded due to technical problems There was a significant increase in temperature in all skin
Ta bl e 2 Temperature response to laser treatment in clipped and unclipped skin, and in underlying muscle, mea-sured 1 and 3 cm from the irradiated area
Area Baseline Treatment Difference Peak value Time to peak
Muscle
Skin 1 cm
clipped (n=7) 30.4±1.0 35.9±0.8* 5.5±1.5 36.9±1.4 175.7±28.8 unclipped (n=8) 29.7±1.3 34.5±1.6* 4.8±1.4† 35.1±1.9 164.0±18.8†
Skin 3 cm
unclipped (n=7) 32.6±0.7 34.7±1.0* 2.1±0.4 35.6±1.1 230.1±21.9
Values are presented as means ±SE; measured as temperature (º C); time to peak (s), n.d.= not detected.*
Significantly different from baseline, † significantly different from unclipped skin 3 cm from irradiated area, p<0.05.
Trang 5recordings, i.e 1 cm and 3 cm from the
irradi-ated area, compared to the respective baseline
recordings, for both clipped and unclipped hair
coat The temperatures did not return to the
pre-treatment baseline at 30 min after pre-treatment in:
2/7 of the horses in the skin 1 cm clipped group,
2/8 in the skin 1 cm unclipped group, 5/8 in the
skin 3 cm clipped group, and 2/7 in the 3 cm
un-clipped group
No significant difference was recorded in
mus-cle temperature for either clipped or unclipped
hair coat The time of peak response to laser
treatment is presented in Table 2 Peak
temper-ature occurred earlier in clipped than in
un-clipped skin measured 3 cm from the irradiated
area In the unclipped groups, peak temperature
occurred later in the 3 cm than in the 1 cm
recording point
Perfusion
The perfusion response to laser treatment is
presented in Table 3 The results presented for
muscle perfusion are from seven (clipped skin)
and six (unclipped skin) horses, since values
less than 3.5 PU were excluded from statistical
analysis There was a significant increase in perfusion in all skin recordings, i.e 1 and 3 cm from the irradiated area for both clipped and unclipped hair coat The perfusion did not re-turn to the pre-treatment baseline 30 min after treatment in: 3/8 of the horses in the 1 cm clipped group, 1/7 in the 1 cm unclipped group, 2/8 in the 3 cm clipped group, and 2/7 in the 3
cm unclipped group There was no significant difference in muscle perfusion
The time for peak response to laser treatment is presented in Table 3 There was no significant difference in time to peak
The control group
Temperature and perfusion data are presented
in Table 4 None or only minor changes were seen in tissue temperature or tissue perfusion in the two horses used as controls
Discussion
In the present study, there was an increase in temperature and perfusion in skin, but not in the underlying muscle Two major findings were identified: (1) treatment with defocused CO2
Ta bl e 3 Perfusion response to laser treatment in clipped and unclipped skin, and in underlying muscle, mea-sured 1 and 3 cm from the irradiated area
Area Baseline Treatment Difference Peak value Time to peak
Muscle
Skin 1 cm
clipped (n=8) 100 434.0±170.5* 334.0±170.5 497.5±182.8 157.3±35.3 unclipped (n=8) 100 363.8±120.2* 263.8±120.2 428.2±139.3 167.2±29.3
Skin 3 cm
clipped (n=8) 100 345.2±123.6* 245.2±123.6 407.9±142.4 184.0±28.4 unclipped (n=8) 100 216.5± 65.3* 116.5± 65.3 263.2± 80.9 208.2±47.1
Values are presented as means ±SE, perfusion (PU) with the baseline set to 100%; time to peak (s), n.d.= not detected * Sig-nificantly different from baseline, p<0.05.
Trang 6laser causes an increase in temperature of the
skin in clipped and unclipped haircoat, 1 and 3
cm from the irradiated area; (2) the increase in
temperature was accompanied by an increased
perfusion To the best of our knowledge, no
study has been performed on the effects on
blood perfusion of defocused CO2laser
treat-ment in horses Therefore, comparisons can
only be made with studies on other modalities
with an effect on tissue temperature and/or
blood perfusion Studies on acupuncture,
tran-scutaneous electric nerve stimulation,
superfi-cial heat and continuous therapeutic ultrasound
have all shown increase in temperature and/or
perfusion (Nannemann 1991, Oosterveld and
Rasker 1994, Cramp et al 2000, Levine et al.
2001, Wright and Sluka 2001, Kuo et al 2004)
Therapeutic application of heat plays a major
role in rehabilitation programs The rationale
for using different heating modalities is based
primarily on the fact that they produce peak
temperatures in different locations The goal is
to achieve a “therapeutic” level of temperature
elevation without causing adverse responses
As one of the explanations for the mode of ac-tion of defocused CO2 is its photothermal ef-fect, it is important to identify the heating pat-tern of laser treatment In the present study, the increases in temperature and perfusion were in superficial tissues, and not in muscle tempera-ture and blood perfusion As in other superficial heating modalities, the deeper tissues including muscles are usually not significantly heated Heat transfer from the skin surface into deeper tissues is inhibited by the subcutaneous fat, which acts as a thermal insulator, and by the in-creased blood flow in more superficial tissues which cools the tissues by transporting away the
heat (Lehmann 1990)
The physiological effect of the applied CO2 laser irradiation is related to the activation of warmth and heat receptors and afferents;
(Arendt-Nielsen and Chen 2003) In the present
study, it is likely that both A␦ and C-fibres were stimulated, with a secondary influence on blood perfusion The mechanism for vasodilatation is suggested to be activation of the axon/dorsal
Ta bl e 4 Temperature and perfusion response in the control horses; temperature and perfusion in clipped and unclipped skin, and underlying muscle, measured 1 and 3 cm from the irradiated area
Area Baseline Treatment Baseline Treatment
Muscle
Skin 1 cm
Skin 3 cm
Values are presented as means/ medians and ranges; measured as temperature (º C); perfusion (PU) with the baseline set to 100%, n=2.
Trang 7root ganglion reflex from heat sensitive
noci-ceptive afferents, which releases
neurotransmit-ters that increase blood flow These
neurotrans-mitters may stimulate nitric oxide release
causing further vasodilatation (Kellogg et al.
1999, Minson et al 2001, Stephens et al 2001).
Thermally evoked vasodilatation has also been
found following non-painful stimulation when
using a slowly increasing heat stimulus (Magerl
and Treede 1996, Minson et al 2001) In the
present study, the perfusion at 1 cm from the
ir-radiated area increased with 146 PU on
aver-age, when the temperature had increased by
about 6 ° C, to a mean of approximately 36 ° C
This is consistent with results from other
stud-ies that report on significant vasodilatation
be-tween local temperatures of 30-35 º C (Barcroft
and Edholm 1943, Taylor et al 1984, Johnson
et al 1986, Stephens et al 2001) In humans,
local warming of the skin to 42° C has been
re-ported to increase blood flow tenfold, at the end
of a 20-min warming period (Saumet et al.
1998)
The increase in perfusion started directly after
the first rise in temperature This is in
agree-ment with an earlier study showing a
correla-tion between the first sensacorrela-tion of non-noxious
heat and the onset of cutaneous vasodilatation,
and that the vasodilatation correlates better
with the sensation of heat compared to actual
skin temperature (Stephens et al 2001) Our
findings, and the fact that vasodilatation was
detected 3 cm from the irradiated site, support
the suggestion that the observed vasodilatation
was caused by an axon/dorsal root ganglion
re-flex of nociceptive afferents, probably in
com-bination with a secondary release of nitric
ox-ide (Kellogg et al 1999, Minson et al 2001)
There were no significant differences between
the temperatures of clipped and unclipped skin
These results do not agree with earlier studies
in which the skin temperature was higher in
ani-mals with long haircoat, compared to short or
clipped hair (Steiss and Adams 1999, Bergh et
al 2005) However, it is possible that the
rela-tively thin haircoat at the actual experimental site had an influence on the results As the
irra-Fi g 1 Representative tracing from one laser treated and one control horse, displaying temperature and perfusion response The perfusion is presented as ar-bitrary Perfusion Units (PU) and temperature as ˚ C Channel 1; perfusion in muscle Channel 2; perfusion
in skin at 1 cm from the irradiated area Channel 3; perfusion in skin at 3 cm from the irradiated area Channel 4; temperature in muscle Channel 5; tem-perature in skin 1 cm from the irradiated area Chan-nel 6; temperature in skin at 3 cm from the irradiated area.
A; one minute tracing immediately before the start of the treatment B; one minute tracing at the end of the treatment C; one minute tracing at four minutes after end of the treatment.
T; start of laser and sham treatment, respectively.
Trang 8diations of the clipped and unclipped areas
were randomized, it is unlikely that a
consen-sual effect of the irradiation would have a major
influence on the results
Movement artefacts are a common problem
us-ing Laser Doppler Flowmetry technique This
was reduced as the horses were anaesthetised
during the study It is possible that tissue
perfu-sion was affected by the anaesthetic agent and
to some extent, by the position of the limb In
order to minimize the negative effects on
pe-ripheral perfusion, the anaesthesia was
main-tained with isoflurane, since hind-limb blood
flow has been found to be higher during
isoflu-rane than halothane anaesthesia, due to a less
cardiac depression and greater peripheral
vas-cular dilatation (Raisis et al 2000a) Blood
flow to a region is influenced by its vertical
po-sition relative to the heart (Hennig et al 1995).
This positional effect was minimized in our
study since the position of the probes was
hori-zontal and approximately at the level of the
heart However, it is likely, due to the influence
of general anaesthesia and positioning of the
limb, that the registered increases in blood
per-fusion in the anaesthetized horses were similar
or less pronounced than would be expected in
non-anaesthetized animals
It has been reported that the surface
tempera-ture of the distal limb differs between
individu-als (Kameya and Yamaoka 1968, Webbon 1978,
Palmer 1983) and it is known that the ambient
temperature has an influence on skin
tempera-ture (Kameya and Yamaoka 1968, Webbon
1978) This variation was greater at an ambient
temperature of 5 ° C than at higher temperatures
(15-25 ° C) (Kameya and Yamaoka 1968,
Palmer 1983) In the present study, the ambient
temperature varied between approximately 16
and 20 ° C
In our study, defocused CO2laser radiation
in-creased temperature and tissue perfusion in the
skin, but not in deeper tissues However, the
question as to whether this has therapeutic sig-nificance remains to be investigated The bio-physical effects of similar temperature eleva-tion in human body tissue include increased local blood flow and metabolism, elevated pain threshold, decreased muscle spindle firing rate, and increased extensibility of connective tissue Heat can provide analgesia, promote relaxation, reduce muscle spasm, and enhance flexibility of
muscles and periarticular structures (Lehmann
1990, Nannemann 1991, Minor and Sanford
1993, Wright and Sluka 2001) Heat also
as-sists in resolution of inflammatory infiltrates,
oedema and exudates (Lehmann 1990, Nanne-mann 1991) Consequently, the increase in
tem-perature and perfusion in the present study may have had an effect on pain and tissue regenera-tion
In conclusion, defocused CO2 laser causes a significant increase in skin perfusion, which is correlated to the increase in skin temperature, both measured at 3 cm from the irradiated area
No differences were observed between clipped and unclipped haircoat, or in muscle Further studies are needed to investigate if the increase
in temperature and perfusion achieved by defo-cused CO2laser enhances tissue regeneration, decreases pain and restores impaired function
Acknowledgements
This project was supported by grants from the Swedish Racing and Totalizator Board (ATG), and EL.EN Srl, Italy supplied the laser system The au-thors express their sincere gratitude to Karin Thulin, Annelie Rydén, Pia Funkquist, Anna Edner, Birgitta Essén-Gustavsson for technical assistance, and Patrik Öhagen for statistical consultation Many thanks also
to Björn Bakken and the late Bertil Gazelius at Per-imed for excellent help with the laser Doppler system.
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Sammanfattning
Lokala blodflödesförändringar hos häst vid behan-dling med defokuserad CO2laser.
Laserbehandling sägs stimulera och påskynda ned-sänktsprocessen, men dess verkningsmekanism är oklar En nyligen publicerad studie visar att behan-dling med defokuserad CO2laser ger en ökning av temperaturen i ytliga vävnader hos häst En ökning
av vävnadstemperatur åtföljs ofta av en ökning av det lokala blodflödet, med en positiv inverkan på väv-naders läkning Så vitt vi vet saknas publicerade studier om defokuserad CO2 lasers effekt på blod-flöde hos häst Syftet med denna studie var att under-söka effekten av defokuserad CO2 laser på lokalt blodflöde (med hjälp av Laser Doppler Flowmetry) och att korrelera blodflödet till temperaturen i rakad och orakad hud, samt i underliggande muskelvävnad Tio hästar inkluderades i studien, varav åtta fick aktiv laser och två placebo Den aktiva laserdosen var 91 J/cm 2 och gavs på ett 42 cm 2 stort område över semimembranosus muskulaturen Den aktiva laser-behandlingen ökade signifikant blodflöde och tem-peratur, med i genomsnitt 146.3±33.4 perfusionsen-heter (334%) och 5.5±1.5 ° C i rakad hud, och 80.6±20.4 perfusionsenheter (264%) och 4.8±1.4 °C
i orakad hud Inga statistiskt signifikanta skillnader kunde noteras i blodflöde och temperatur i underlig-gande muskel, eller mellan rakad och orakad hud Fortsatta studier får visa om denna temperatur- och blodflödesökning kan leda till smärtlindring och för-bättrad läkning
(Received October 26, 2005; accepted November 14, 2005).
Reprints may be obtained from: A Bergh, Swedish University of Agricultural Sciences, Department of Anatomy and Physiologi, P.O Box 7011, SE-750 07 Uppsala, Sweden.